This invention relates to a new antimicrobial compound, WF14573 or its salt. More particularly, it relates to a new antimicrobial compound, WF14573 or its salt which has an antimicrobial activity against pathogenic microorganisms, especially pathogenic fungi, a process for the preparation thereof and a pharmaceutical composition comprising the same.
The new compound, WF14573 is represented by the following formula: 
wherein R1 is hydrogen or methyl and R2 is hydrogen or palmitoyl.
In this specification, the following designations of the specific compound are conveniently used.
The new compound, WF14573A has the following physico-chemical properties:
a) Molecular weight: ESI-MS(negative)m/z 1143 (Mxe2x88x92H); b) Elemental analysis: C 48.39; H 7.15; N 8.95; c) Melting point: 230-240xc2x0 C. (dec.); d) Optical rotation: [xcex1]D23=xe2x88x9212 (c 0.5, methanol); e) UV spectrum: xcex max(xcex5)=276 nm (methanol); f) IR spectrum: xcexd max(KBr)=3360, 2920, 2830, 1670, 1630, 1540, 1440, 1270, 1240, 1050 cmxe2x88x921; g) 1H-NMR spectrum: (500 MHz, CD3OD) xcex4 (ppm): 7.18 (1H, d, J=8 Hz), 6.80 (1H, d, J=2 Hz), 6.67 (1H, dd, J=8, 2 Hz), 5.30 (1H, d, J=3 Hz), 5.10 (1H, d, J=4 Hz), 4.98 (1H, d, J=3 Hz), 4.64 (1H, m), 4.58xcx9c4.52 (3H, m), 4.47xcx9c4.43 (2H, m), 4.40xcx9c4.33(2H, m), 4.27 (1H, d, J=3 Hz), 4.02xcx9c3.95 (3H, m), 3.82xcx9c3.76 (2H, m), 2.70 (1H, m), 2.63xcx9c2.55 (2H, m), 2.48xcx9c2.40 (2H, m), 2.30xcx9c2.17 (3H, m), 2.05xcx9c1.93(4H, 1.57 (2H, m), 1.35xcx9c1.23 (24H, m), 1.15 (3H, d, J=6 Hz), 0.89 (3H, t, J=7 Hz). h) 13C-NMR spectrum: (125 MHz, CD3OD) xcex4 (ppm): 176.8 (s), 175.9 (s), 174.4 (s), 174.1 (s), 172.9 (s), 172.7 (s), 172.5 (s), 169.3 (s), 150.3 (s), 140.1 (s), 137.6 (s), 124.0 (d), 121.9 (d), 119.4 (d), 74.2 (d), 74.0 (d), 73.9 (d), 71.3 (d), 70.7 (d), 70.5 (d), 69.8 (d), 68.3(d) 62.4 (d), 58.5 (d), 58.0 (d), 57.2 (t), 55.4 (d), 51.2 (d), 47.0 (t), 41.1 (t), 39.7 (t), 38.9 (t), 36.7 (t), 35.0 (t), 34.6 (t), 33.1(t), 30.80 (txc3x975), 30.76 (t), 30.7 (t), 30.6 (t), 30.5 (t), 30.3 (t), 27.0 (t). 23.7 (t), 19.5 (q), 14.4 (q). i) Solubility; Soluble: water, methanol. dimethylsulfoxide; Insoluble: n-hexane, chloroform; j) Thin layer chromatography: Carrier: Silica gel 60 F254 (Merck); Solvent: 1-butanol: acetic acid: water=4:1:2; Rf=0.39.
The new compound, WF14573B (as its sodium salt), has the following physico-chemical properties:
a) Molecular weight: ESI-MS(negative)m/z 1157 (Mxe2x88x92H); b)Elemental analysis: C 48.79; H 7.34; N 8.96; S 2.80; Na 1.69; c) Melting point: 220-225xc2x0 C. (dec.); d)Optical rotation: [xcex1]D23=15xc2x0 (c 0.9, methanol); e) UV spectrum: xcex max(xcex5)=276 nm (methanol); f) IR spectrum: xcexd max(KBr)=3360, 2940, 2830, 1670, 1630, 1530, 1440, 1270, 1240, 1050 cmxe2x88x921; g) 1H-NMR spectrum: (500 MHz, CD3OD) xcex4 (ppm): 7.18 (1H, d, J=8 Hz), 6.80 (1H, d, J=2 Hz), 6.67 (1H, dd, J=8, 2 Hz), 5.29 (1H, d, J=3 Hz), 5.07 (1H, d, J=4 Hz), 4.99 (1H, d, J=4 Hz), 4.64 (1H, m), 4.58xcx9c4.51 (3H, m), 4.48xcx9c4.36 (4H, m), 4.16 (1H, m), 4.07 (1H, m), 4.02xcx9c3.97 (2H, m), 3.79 (1H, br. d, J=11 Hz), 3.37 (1H, m), 2.69 (1H, m), 2.60xcx9c2.40 (5H, m), 2.22 (2H, m), 2.05xcx9c1.98 (3H, m), 1.57 (2H, m), 1.35xcx9c1.24 (24H, m), 1.15 (3H, d, J=6 Hz),1.06 (3H, d, J=7 Hz), 0.89 (3H, t, J=7 Hz). h) 13C-NMR spectrum: (125 MHz, CD3OD) xcex4 (ppm): 176.7 (s), 176.0 (s), 174.3 (s), 174.1 (s), 172.8 (s), 172.5 (s), 172.5 (s), 169.3 (s), 150.3 (s), 140.1 (s), 137.6 (s), 124.0 (d), 122.0 (d), 119.4 (d), 75.9 (d), 74.0 (d), 73.9 (d), 71.3 (d), 70.7 (d), 70.5 (d), 70.2 (d), 68.2 (d), 62.4 (d), 58.6 (d), 58.0 (d), 57.2 (t), 55.5 (d), 52.9 (t), 51.3 (d), 41.1 (t), 39.8 (t), 39.1 (d), 38.9 (t), 36.7 (t), 34.9 (t), 33.0 (t), 30.8 (txc3x975), 30.7 (t), 30.7 (t), 30.5 (t), 30.4 (t), 30.3 (t), 27.0 (t), 23.7 (t), 19.5 (q), 14.4 (q), 11.1 (q). i) Solubility; Soluble: water, methanol, dimethylsulfoxide; Insoluble: n-hexane, chloroform; j) Thin layer chromatography: Carrier: Silica gel 60 F254 (Merck); Solvent: 1-butanol: acetic acid: water=4:1:2; Rf=0.45.
In the result of the above physicochemical properties and extensive investigations, the chemical structures of WF14573A and B are identified as mentioned above, respectively.
According to this invention, the compound, WF14573A and B can be prepared by culturing a WF14573A and/or B-producing strain belonging to the genus Coleophoma in a nutrient medium.
Particulars of microorganisms used for the production of WF14573A and B and production thereof will be explained in the followings.
Microorganism
The microorganism which can be used for the production of WF14573A and B is a WF14573A and/or B-producing strain belonging to the genus Coleophoma, among which Coleophoma empetri No.14573 was newly isolated from a decayed leaf sample, collected at Mitsushima-cho, Kamiagata-gun, Nagasaki-ken, Japan.
Lyophilized samples of the newly isolated microorganism, Coleophoma empetri No. 14573 was deposited with an International Depository Authority on the Budapest Treaty, National Institute of Bioscience and Human-Technology, 1-3. Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305, Japan under the deposit number FERM BP-6252 on Feb. 12, 1998.
It is to be understood that the production of the new compound, WF14573A and/or B is not limited to the use of the particular organism described herein, which is given for illustrative purpose only. This invention also includes the use of any mutants which are capable of producing the WF14573A and/or B including natural mutants as well as artificial mutants which can be produced from the described organism by conventional means, such as genetic engineering, X-ray, ultraviolet radiation, treatment with N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine and the like.
The strain No.14573 has the following morphological, cultural and physiological characteristics.
The strain grew rather restrictedly on various culture media, and formed grayish colonies. The strain produced pycnidial to stromatic conidiomata on the autoclaved leaf segments affixed on agar media, while it formed neither teleomorph nor anamorph on or in the media. The conidiomata were convex to discoid, dark brown to black, and formed ampulliform to lageniform conidiogenous cells on the lower cells of their inner walls. Conidia were hyaline, one-celled and cylindrical. On the basis of comparing the morphological characteristics with fungal taxonomic criteria by von Arx (J. A. von Arx: The Genera of Fungixe2x80x94Sporulating in Pure Culture. 3rd ed., pp.315, J. Cramer, Vaduz, 1974) and by Sutton (B. C. Sutton: The Coelomycetesxe2x80x94Fungi Imperfecti with Pycnidia, Acervuli and Stroma., pp.696, Commonwealth Mycological Institute, Kew, 1980), strain No.14573 was considered to belong to the coelomycete genus Coleophoma Hxc3x6hn. 1907 (Sphaeropsidales). Its mycological characteristics were as follows.
Cultural characteristics on various agar media are summarized in Table 1. Culture on potato dextrose agar grew restrictedly, attaining 1.5-2.5 cm in diameter two weeks later at 25xc2x0 C. This colony surface was plane to raised, felty to cottony, light gray to dark gray, and yellowish white at the margin. The reverse color was olive. Conidial structures were not observed. Colonies on corn meal agar grew rather restrictedly, attaining 2.5-3.5 cm in diameter under the same conditions. The surface was plane to centrally raised, dark gray and lustrous. At the colony center was felty to cottony and purplish gray to dark purple. Mycelium near the margin was submerged and white. The reverse was dark gray to dark green, and vellowish white at the margin. Conidial structures were not produced.
The morphological characteristics were determined from the cultures on the sterile leaf segments affixed on a Miura""s LCA plate (Miura, K. and M. Kudo: Trans. Mycol. Soc. Japan. 11:116-118, 1970). Conidiomata formed on the leaf segments alone. They were pycnidial to stromatic, superficial to semi-immersed, separate and dark brown to black. Their shape was convex to discoid, sometimes papillate, non-ostiolate or indistinctly ostiolate, unilocular, flattened at the base, thin-walled at the upper part, 70-170 xcexcm in diameter and 40-90 xcexcm high. The lower cells of inner pycnidial walls were thick-walled, dark brown, irregularly shaped, and formed textura angularis. The inner cells produced directly conidiogenous cells, but they sometimes formed conidiophores. The conidiophores were hyaline, smooth, septate, simple to sparingly branched, and 10-17xc3x973.5-4.5 xcexcm. The conidiogenous cells were discrete, acrogenous or intercalary, hyaline, smooth, ampulliform to lageniform, sometimes cylindrical, and 5-11(-16)xc3x972-4.5 xcexcm. Conidia were holoblastic, hyaline, smooth, one-celled, cylindrical, rounded at the apical end, with a small projection at the base, and (11-)13-20xc3x972-3 xcexcm. Paraphyses were often formed on or amongst conidiophores, and their structures were similar to sheaths covering with conidiogenous cells and conidia. They were hyaline, thin-walled, campanulate to cylindrical, collapsing at later stage, and 18-30(-35)xc3x972.5-5 xcexcm. Vegetative hyphae were smooth, septate, brown and branched. The hyphal cells were cylindrical and 2-7 xcexcm in width. Chlamydospores were not observed.
Strain No.14573 was able to grow at the temperature range from 3 to 30xc2x0 C., with the growth optimum at 21 to 24xc2x0 C. These temperature data were determined on potato dextrose agar (made by NISSUI).
According to the taxonomic criteria of the genus Coleophoma by Wu et al. (W. Wu, B. C. Sutton and A. C. Gange: Mycol. Res. 100: 943-947. 1996.), the strain No.14573 resembles Coleophoma empetri (Rostr.) Petrak 1929. There were few differences between above characteristics and this species description: superficial and indistinctly ostiolate conidiomata. Added to these, it remains one question for the structures described as paraphyses. Much more observation was needed to determine the conidial ontogeny of this genus. In conclusion, we identified the isolate as one strain of Coleophoma empetri, and named it Coleophoma empetri No. 14573.
These characteristics were observed after 14 days of incubation at 25xc2x0 C. The color descriptions were based on Methuen Handbook of Colour (Kornerup, A. and J. H. Wanscher, 3rd ed., pp.252., Methuen, London, 1978).
Production of WF14573A and B
The compound, WF14573A and B can be prepared by culturing a WF14573A and/or B-producing strain in a nutrient medium.
In general, WF14573A and B can be produced by culturing the WF14573A and/or B-producing strain in a nutrient medium containing assimilable sources of carbon and nitrogen, preferably under aerobic conditions (e.g. shaking culture. submerged culture, etc.).
The preferred sources of carbon are carbohydrates such as sucrose, glucose, soluble starch and the like.
The preferred sources of nitrogen are cottonseed meal, soybean flour, yeast extract, peptone, gluten meal, corn steep liquor, dried yeast etc. as well as inorganic and organic nitrogen compounds such as ammonium salts (e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea, amino acid and the like.
The carbon and nitrogen sources need not be used in their pure form, because less pure materials which contain traces of growth factors and considerable quantities of mineral nutrients, are also suitable for use. Further, there may be added to the medium. Mineral salts such as calcium carbonate, sodium or potassium phosphate magnesium salts and the like. If the culture medium is foamed remarkably, a defoaming agent such as liquid paraffin, higher alcohol, plant oil, mineral oil and silicones may be added.
Preferred production conditions of WF14573A and/or B in massive amount may includes a submerged aerobic cultural condition.
Preferred production conditions of WF14573A and/or B in small amount may include a shaking or surface culture in flask or bottle.
In case where the production is carried out in a large tank, it is preferable to use the vegetative form of the organism for inoculation in the production tank in order to avoid growth lag.
Agitation and aeration of the culture broth may be accomplished in a variety of ways. Agitation may be provided by a propeller or the similar mechanical agitation equipment, by revolving or shaking the fermentor, by various pumping equipment or by the passage of sterile air through the medium.
The fermentation is usually conducted at a temperature between 20xc2x0 C. and 35xc2x0 C., preferably about 25xc2x0 C. for 50 to 100 hours, which may be varied depending on the fermentation condition and scale.
Thus produced WF14573A and B can be recovered from the cultured broth by conventional means which are commonly used for the recovery of other fermentation products such as antibiotics.
In general, most of the WF14573A and B produced are found in the culture filtrate as well as in the cells. Accordingly, WF14573A and B can be isolated from the filtrate and the cells, which is obtained by filtering or centrifuging the broth, by a conventional method such as concentration under reduced pressure, lyophilization, extraction with a solvent, pH adjustment, treatment with a resin (e.g. anion or cation exchange resin, non-ionic adsorption resin), treatment with an adsorbent (e.g. activated charcoal, silicic acid, silica gel, cellulose, alumina), crystallization, recrystallization and the like. The WF14573A and B obtained in its free form may also be converted to its salts by treating WF14573A and B with an inorganic or organic base such as sodium or potassium hydroxide, ammonium hydroxide, ethanolamine and the like and with an amino acid such as glycine, lysine, glutamic acid and the like.
The WF14573 and B have a strong antimicrobial activity against pathogenic microorganisms, especially pathogenic fungi such as pathogenic yeast (e.g. Candida albicans etc.) and the like. Accordingly, the WF14573A and B and their pharmaceutically acceptable salt are useful as an antimicrobial agent, especially antifungal agent which is used for the treatment of infectious diseases in human beings and animals.
As examples for showing such pharmacological effects of WF14573A and B, some pharmacological test data are illustrated in the followings.
Test 1 (Antimicrobial Activity)
Antimicrobial activity of WF14573A or B was determined by a serial broth dilution method using 96-well microtiter plate in 100 xcexcl of veast nitrogen base dextrose medium. The inoculum was adjusted to 1xc3x97105 colony forming units/ml. Candida albicans and Aspergillus fumigatus were cultured at 37xc2x0 C. for 24 hours and Cyptococcus neoformans was cultured at 37xc2x0 C. for 48 hours in 5% CO2 incubator. After incubation, the growth inhibition of microorganism in each well was determined by microscopic observation. The results were shown as MEC (minimum effective concentration: xcexcg/ml) value (Table 1).
The deacyl WF14573A or B or their salts can be prepared by deacylating WF14573A or B or their salts, especially by deacylating WF14573A or B or their salts respectively, in the presence of cultured broth or its processed material of a microoranism [such as a microorganism belonging to the genus Streptomyces, e.g. Streptomyces anulatus No. 4811 (FERM BP-5808)] which is capable of deacylating WF14573A or B to give deacyl WF14573A or B.
The processed material of the cultured broth may include mycelia and crude or purified deacylase preparations obtained from them.
The enzymatic reaction is carried out in a conventional manner, e.g. those described in the following working Examples.
The deacyl WF14573A or B can be converted to an acyl derivative thereof by a conventional acylation. The acyl derivative can be represented by the above formula (I) wherein R2 is acyl except palmitoyl.
The present antimicrobial agent comprising the WF14573 (I) or pharmaceutically acceptable salt thereof is useful as a therapeutic agent for infectious diseases in animals including human beings. The pharmaceutically acceptable salt of the WF14573 (I) may include the salt as exemplified above.
The antimicrobial composition can be used in the form of pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains the WF14573 (I) or its salt in admixture with a pharmaceutical organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications. The active ingredient may be compounded, for example, with usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, ointments and any other form suitable for use. The pharmaceutically acceptable carriers are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for use in manufacturing preparations and in addition, auxiliary, stabilizing, thickening and coloring agents and perfumes. The antimicrobial compositions can also contain preserving or bacteriostatic agents thereby keeping the active ingredient in the desired preparations stable in activity. The active object compound is contained in the antimicrobial composition in an amount sufficient to produce the desired therapeutic effect upon the bacterially infected process or condition.
For applying this composition to human patients, it is preferably to apply it in a form of intraveneous, intramuscular, oral or percutaneous administration. While the dosage or therapeutically effective amount of the WF14573 (I) or pharmaceutically acceptable salts thereof varies depending on the age, conditions of each individual patient to be treated, the preferred daily dosage of the WF14573 (I) can be selected from the range of 0.1-100 mg/kg of the patient.